Morphofunctional changes in the midgut of tick females of the genus Ixodes (Acari: Ixodidae) during and after feeding

Cyclic changes of the midgut epithelium were observed in females of 5 ticks species of the genus Ixodes during 7-10 days of feeding. The midgut epithelium of unfed females is represented by the digestive cells of nymphal phase and stem cells. The digestive cells of nymphal phase are functional during 1.5-2 days after attachment of the tick, and then, after the tearing away they go into the gut lumen. The secretory cells substitute the digestive cells of nymphal phase and finish their growth during the 4-4.5 days. Secretion of digestive enzymes is performed by the holocrine type with tearing away a whole cell. Intracellular digestion takes place in the digestive cells of four consequent generations. The secretory and digestive cells form a peritrophic matrix on their surface. The presence of peritrophic matrix gives an evidence the maturity and functional activity of the secretory and digestive cells. We suggest, that the peritrophic matrix takes part in intracellular digestion, namely in the process of micropinocytosis. The phagocytosis was not found in the ticks investigated. Digestion in the midgut lumen is performed by enzymes of the ruptured secretory and digestive cells, that is proved by the haemolysis of erythrocytes in the zone of their contact with these cells. The digestive cells of each generation functioned almost synchronously, with largest difference in starting about 12 hours.     

Specificity and effects of host resistance on the African tickRhipicephalus zambeziensis (Acarina: Ixodidae)

Repeated infestations of the rabbit with adultRhipicephalus zambeziensis ticks resulted in a tenfold reduction in engorgement weight of adult ticks and a significant decline in engorgement weight of nymphs. There was no evidence of cross-resistance against the South African bont tick,Amblyomma hebraeum. Immunization of rabbits with crude extracts of whole-tick protein significantly influenced engorgement weights and oviposition rates of adultR. zambeziensis.Histological studies of tissues fromR. zambeziensis adults fed on resistant rabbits showed severe gut damage; this was associated with the binding of host IgG to mid-gut cells.     

Ultrastructure of the midgut and blood meal digestion in the adult tickDermacentor variabilis

Digestive cells in the midgut of male and femaleDermacentor variabilis (Say) took up the blood meal in coated vesicles and smooth flask-shaped vesicles, and deposited it in endosomes which were digested via heterophagy. Iron was concentrated in residual bodies.Digestion occurred in three distinct phases in mated females: (1) continuous digestion (initiated by feeding) occurred during slow engorgement; (2) reduced digestion (initiated by mating) occurred in mated females during the period of rapid engorgement; (3) a second continuous digestion phase (initiated by detachment from the host) occurred throughout the post-feeding periods of preoviposition and oviposition.It is proposed that the stem cells in the midguts of unfed females were progenitors of digestive, replacement, and presumed vitellogenic cells in midguts of mated feeding females. Digestive cells were present in all three digestion phases. Only during the first continuous digestion phase did digestive cells fill up with residual bodies, rupture and slough into the lumen, or did whole cells slough into the lumen. During the other two digestion phases no sloughing of digestive cells was observed. At the end of oviposition the digestive cells were filled with residual bodies. Replacement cells were present only during the first continuous-digestion phase. Presumed vitellogenic cells were present only during the reduced-digestion phase and during the second continuous-digestion phase. Stem cells in unfed males developed only into digestive cells in feeding males. Fed males and fed unmated females had only the first continuous-digestion phase. After being hand-detached from the host, unmated 13-day-fed females went through cellular changes associated with the reduced-digestion phase and second continuous-digestion phase of fed mated females, then began ovipositing. Maximum development of the basal labyrinth system and lateral spaces matched the known time of maximum water and ion movement across the midgut epithelia.Spectrophotometric analyses of lumen contents and midgut cells, sampled after detachment from the host, showed that concentrations of protein and hemoglobin at day 1 post-detachment decreased by one-half at the beginning of oviposition, while hematin increased about twofold by the end of oviposition. This supported the idea of the presence of a second continuous-digestion phase.     

Immunisation of rabbits against the brown ear tick,Rhipicephalus appendiculatus using tick haemolymph

Tick naive rabbits were immunised with haemolymph components from partially fed Rhipicephalus appendiculatus adult ticks and subsequently challenged with all the developmental instars of the tick. The results obtained showed that the rabbits were rendered resistant to all the instars of the tick. However, the resistance was more pronounced in adult ticks than in the immature stages. The resistance was manifested as a reduction in the number of ticks that fed successfully to engorgement, reduced engorgement weights and reduced fecundities. Re challenging the resistant experimental rabbits with all the developmental instar stages of Rhipicephalus appendiculatus showed that resistance was maintained in subsequent infestations.     

Artificial immunization of rabbits with Hyalomma dromedarii tick-derived midgut antigen

New Zealand white rabbits were immunized with partly fed Hyalomma dromedarii tick-derived midgut concealed antigens (supernate and pellet fractions) and Freund's complete adjuvant (FCA). The rabbits received three inoculations subcutaneously on days 0, 14 day 21 at a dose rate of 1 mg antigen per animal. The effects of the immunity induced was determined by infesting the rabbits with adult H. dromedarii ticks. In immunized rabbits a significant reduction in tick yield, engorgement weight, oviposition period, egg mass weight and percentage of egg hatchability was found. The gut supernatant antigen fraction induced the best protection in terms of reduced feeding and reproductive performance of the ticks.     

Histological study of the attachment sites of adult Rhipicephalus appendiculatus on rabbits and cattle

Biopsies of tick attachment sites on ears of five rabbits and four calves (Bos taurus) were taken at first and third infestations and were examined by histochemistry, light microscopy and electron microscopy. Resistance was expressed by all hosts by reduction of tick engorgement weights. Attachment sites at first infestations on rabbits and cattle were similar and were characterized by an acute inflammatory abscess with a preponderance of neutrophils and macrophages infiltrating the site. Attachment sites at third infestations on rabbits and cattle were similar and were characterized by infiltration of neutrophils and macrophages and a 2- to 5-fold increase in the proportions of eosinophils and basophils. Lymphoblasts and plasmacytes were found in third infestation sites. In rabbits there was much necrosis and in cattle there were large intraepidermal pustules in third infestations. The cement attachment cone of the ticks was of lipoprotein and contained aminopeptidase. Salivary glycoproteins and esterases were detected in the attachment sites.     

Feeding performance ofAmblyomma variegatum (Acarina: Ixodidae) fed repeatedly on rabbits

Rabbits infested with different stages ofAmblyomma variegatum Fabricius, 1794 became resistant to subsequent infestations by the same tick. Resistance was manifested by a reduction of 73.6% in the mean engorged weight of female ticks by the 3rd infestation. By the 5th infestation, only 70% of the nymphs engorged and their mean engorged weight was 57.7% of that of nymphs fed on tick-naive control rabbits. In the case of larvae, there was a 22.8% reduction in their mean engorged weight by the 6th infestation. Based on increases in body length while feeding on tick-naive rabbits, the nymphs could be divided into four feeding categories, i.e. Nl (unfed), N2, N3 and N4 (increasing states of engorgement). While 92–96% of ticks which dropped from susceptible rabbits fitted into feeding category N4, only 28% of those that dropped from tick-resistant rabbits fitted into that category. The remainder of the ticks fitted into categories N3 (60%) and N2 (12%). The majority of those ticks with reduced weight developed into adults which had an atypical scutal ornamentation pattern, in that an additional pair of lateral spots was regularly observed on the males. The females of such ticks deposited small numbers of eggs from which no larvae hatched.     

Digestion in the cattle-tick Boophilus microplus: light microscope study of the gut cells in nymphs and females

The gut caeca of B. microplus were studied by light microscopy using paraffin and methacrylate embedded material. It has been shown that during feeding of nymphs and adults, the midgut consists of five cell types, stem cell, digest cell, secretory cells (s₁) and (s₂) and basophilic cell. The stem cell differentiates into any of the other cell types. The digest cell matures through a series of stages and has up to three generations during feeding on the host. The final generation has two distinct cell types, the first type is thought to be capable of both phagocytosis and pinocytosis. Cells of the second type are predominant at the end of feeding, and may be specialized to ingest and digest haemoglobin. The final stage of the digest series is the spent digest cell which discharges its content into the gut lumen or is excreted whole. The basophilic cell has structures which suggest that one of its functions is to transport digested materials, water and ions across the gut. Secretory cell (s₁) secretes a glycoprotein which may be a haemolysin and secretory cell (s₂) secretes the gut “colloid” mass, an acid mucopolysaccharide, which may function as an anticoagulant. Intracellular digestion leads to the breakdown of host blood and storage of lipid and glycogen in the digest cells.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Population regulation in ticks: the role of acquired resistance in natural and unnatural hosts

Attachment, engorgement and subsequent development of successive infestations of Ixodes trianguliceps larvae and nymphs on natural hosts, Apodemus sylvaticus, and unnatural hosts, laboratory mice, are compared. In laboratory mice, primary infestations above a threshold level of about 10 ticks elicit an immunological response which reduces, in a density-dependent manner, the rate of successful tick engorgement during subsequent infestations. In contrast, in A. sylvaticus successive infestations of larvae result in unchanged or slightly improved survival through to nymphs. The relevance of these results to the concept of host-parasite co-evolution and to tick population regulation is discussed.     

Ultrastructural studies on the midgut epithelium and digestion in the female tickArgas (Persicargas) arboreus (Ixodoidea: Argasidae)

The ultrastructure of the midgut epithelium and digestion in the female tickArgas (Persicargas) arboreus are described before and after feeding, up to oviposition. The epithelium consists of secretory cells, digestive cells (DI and DII), and regenerative cells which may differentiate into any of the other cell types. In unfed ticks, the midgut wall consists mainly of type DII digestive cells retained from a previous feeding, and a few regenerative cells. Within 3 days after the tick feeding, haemolysis of the host blood components occurs in the midgut lumen. Secretory cells, the first differentiation of the regenerative cells, are presumed to produce a haemolysin and an anticoagulant which are released by merocrine and holocrine secretions. The DII cells seen in unfed ticks, and secretory cells which have completed their secretory cycle, start to have a specialized surface for endocytosis characteristic of type DI digestive cells. From 5 to 7 days after feeding up to the female oviposition, type DI cells which have completed their endocytosis are transformed into type DII digestive cells specialized for intracellular digestion and the storage of reserve nutrients required by the tick for long starvation. The various phases of the digestive cycle are considered according to ultrastructural changes of the midgut epithelium.     

Hyalomma anatolicum anatolicum: the role of humoral factors in the acquisition of host resistance

A significant degree of resistance to Hyalomma anatolicum anatolicum can be adoptively transferred to naive recipients with immune serum from rabbits repeatedly infected with adult H. a. anatolicum. Ticks fed on recipients of immune serum took longer to become engorged and showed a significant decrease (P less than 0.01) in engorgement weight and oviposition compared with ticks that fed on recipients of normal serum. A direct correlation between resistance and anti-saliva IgG antibodies was indicated by a progressive increase in the degree of resistance and IgG antibody titres following successive tick infestations. Challenge feeding sites on actively sensitised hosts and recipients of immune serum revealed significantly greater infiltration of basophils and eosinophils compared with feeding sites on recipients of normal serum. However, both the degree of resistance and the accompanying cutaneous basophil and eosinophil responses in recipients of immune serum were considerably weaker than those induced by active tick feeding, thus suggesting that nonhumoral (cell-mediated) mechanisms might also be involved in acquired host resistance to H. a. anatolicum.     

Development of acquired resistance precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodoidea: Ixodidae).

The resistance to the ixodid tick, Haemaphysalis longicornis (parthenogenetic Okayama strain), manifested as a reduction in engorged body weight, developed in rabbits subjected to a series of adult female infestations. A single infestation with females always produced resistance in hosts. This production appeared to depend little on the number of ticks per infestation. Unlike the previous papers, this study revealed that there was no reduction in the mean recovery rate of engorged females when ticks fed on a rabbit repeatedly infested with the ticks. A series of infestations were carried out comparatively to investigate the major biological characters of ticks, such as feeding, oviposition, and hatchability of eggs. As a result, there were no marked differences in these characters among the infestations. Especially, no differences were noticed in the concentration of ingested blood meal in detached females among the infestations. Precipitating antibodies were found in the sera of rabbits resistant to the tick-bite. They were subjected to fractionation by Sephadex G-200 chromatography and tested for sensitivity to 2-mercaptoethanol. As a result, they were proved to be of immunoglobulin of 7 S class.     

Female tick Hyalomma marginatum marginatum salivary glands: preliminary study on protein changes during feeding process and antigens recognized by repeatedly infested cattle

Proteins extracted from salivary glands of unfed, three days and five days fed adult Hyalomma marginatum marginatum were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We have noticed changes during the three feeding steps. Some proteins disappeared during feeding process (23, 38, 39, 40 to 50, 95 and 112 kDa), they might be proteins which were converted in other substances and are secreted. Other antigens (13 to 14, 20, 25, 29, 165 and 210 kDa) were synthesized as a result of tick attachment and feeding. They may be related to growth and development or are the ciment which fixed the adult. Also, three Holstein calves were infested five times with 100 pairs of adult ticks of the same species. The five infestations were performed two weeks from the previous infestation. The sera before infestations and after each infestation were used in western-blot analyses to identify antigens from five days salivary gland extracts of the primary infestation of ticks. Three antigens (18.7, 50 and 80 kDa) were revealed weakly after the first and the second infestations by sera samples but not at infestation onward. Others (13.5, 17 to 18.5, 25, 30, 70, 133, 176 and 193 kDa) were revealed only by sera taken after manifestation of resistance (third infestation). A 13.5 kDa antigen was particularly revealed when resistance had appeared and became more evident after the fourth and fifth infestations. The late antigens recognized might be associated with establishment of calves resistance against ticks.     

The effects of tick numbers and intervals between infestations on the resistance acquired by guinea-pigs to adults of Rhipicephalus evertsi evertsi (Acari: Ixodidae)

Guinea-pigs were infested three times at short (1 week) intervals or long (10 week) intervals between successive infestations with low (1), medium (8) and high (15) numbers of adult Rhipicephalus evertsi evertsi. To compare the resistance developed by the hosts at short and long intervals between infestations, a challenge (fourth) infestation was performed with the high number of ticks. Resistance was assessed by comparing the mean weight of engorged female ticks that dropped from the hosts. Concentrations of beta globulins in the host sera were also monitored. Different tick Ioads (low, medium and high) and infestation intervals had a similar influence on the reduction in weight of the females which was between 60–70% after the final challenge. However, the concentration of beta globulins, was highest in the guinea-pigs exposed to medium and high numbers of ticks with long infestation intervals, probably because of an increase in the production of protective antibodies in response to more intensive antigenic challenge. The relationship between engorgement weight and beta globulin levels in host sera is discussed.     

The fine structure of the digestive tubules of the Marine Bivalve Cardium edule

The epithelium lining the digestive tubules of Cardium edule consists of three cell types, namely mature digestive cells, mature secretory cells and immature flagellated cells. Both the secretory and flagellated cells exhibit a pronounced basiphilia and occur in well-defined crypts. The secretory cells are pyramidal in shape and characterized by the possession of a well-developed granular endoplasmic reticulum and Golgi apparatus. Golgi vesicles derived from the latter migrate to the apical region of the cell where they release their contents into the lumen of the tubules. It is possible that the secretion contains enzymes and although it is likely that such enzymes would function primarily in the lumen of the tubules they may also be the source of the weak proteolytic activity which has been recorded in the gastric fluid of many bivalves. The immature flagellated cells are columnar in shape and possess a poorly developed endoplasmic reticulum and numerous free ribosomes. Although no evidence for this was obtained it is suggested that they may serve to replace either or both of the mature cell types. The digestive cells vary from cuboidal to columnar, possess distinctive Golgi elements with characteristic intracisternal membranous elements, and are capable of ingesting exogenous material from the lumen of the tubule. The process of ingestion was examined following feeding experiments with (a) a mixture of iron oxide and colloidal graphite (Aquadag), (b) whole blood from pigeon and (c) ferritin. Individual particles of graphite were enclosed in phagosomes by a process of phagocytosis, while the proteins haemoglobin and ferritin were ingested by a process of pinocytosis; the membrane enclosing the pinocytic vesicles possesses a characteristic outer granular coat. The contents of both the phagocytic and pinocytic vesicles were transferred to larger bodies considered to be primarily phagosomes in the sub-apical regions of the cell. These possess an interconnecting system of membrane-bound channels which ramifies through the apical cytoplasm. Phagolysosomes deeper in the cytoplasm of the cell were identified by the presence of exogenous material and a positive reaction to tests for acid phosphatase activity. They showed changes in appearance which could be put into a series suggestive of the progressive intracellular digestion of the ingested material.     

Observations on Antricola ticks: small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccessful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gut.     

Ultrastructural changes in the abdominal midgut of the mosquito, Culiseta melanura, during the gonotrophic cycle

Abdominal midguts of the mosquito, Culiseta melanura, were examined by light and electron microscopy 1 hr-14 days days after blood feeding. Epithelial cells were drastically altered from columnar to squamous in form after engorgement, and returned to columnar by day 4 after feeding. Accumulation of mitochondria along brush borders of digestive cells, followed by the appearance of large secondary lysosomes, accompanied blood digestion. Evidence was obtained that myelin-like material in the lysosomes, probably the result of mitochondrial autolysis, is extruded into the lumen. Digestive cells resumed their pre-blood meal appearance by 10-14 days post-engorgement. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. Other midgut cells containing large, microvilli-lined apical cavities were identified in most specimens. No evidence of division or differentiation was obtained for any cell types.     

Schistosoma mansoni: uptake of exogenous hemeproteins by schistosomules grown in vitro

The uptake and fate of the hemeproteins horseradish peroxidase (HRPO) and hemoglobin (Hb) by schistosomules of Schistosoma mansoni maintained in vitro were studied by electron microscopy and cytochemical techniques. After administration of HRPO, reaction product was observed initially in the lumen of the digestive tract, and, after 2 hr of feeding, reaction product was also visible in the cytoplasm of the gastrodermis. There was no evidence of pinocytosis. After administration of Hb, reaction product was observed only in the lumen of the digestive tract. As is found following red blood cell feeding, digestive pigment was formed in the lumen of the gut following Hb feeding. The possible significance of these findings is discussed.     

C3 levels in the sera of rabbits infested and reinfested withIxodes ricinus L. and in midguts of fed ticks

Serum C3 levels of rabbits infested 3 times withIxodes ricinus L. females and C3 in midgut extracts of fed ticks have been measured by a single radial immunodiffusion test.From the first tick attachment, the mean serum C3 level of hosts increased. A peak of C3 occurred 6 days after the beginning of each infestation and was highest during the third infestation (about 8 times more pronounced than the level measured before the first). After the end of all infestations of the rabbits, the serum C3 level decreased and approached normal levels.Modifications of serum C3 levels during reinfestations influenced C3 contents in the blood meal of fed ticks. Midguts of ticks fed during the third infestation contained more C3 than midguts of ticks of the same engorged weights fed during the first or second infestations.